The present invention relates generally to the field of analysis, for example biological analysis. More specifically, the present invention relates to a method for detecting and directly identifying at least one microorganism by an optical route in an, optionally enriched, biological sample.
Microbiological analysis requires accurate methods, in which the time to obtain the result must be as short as possible.
In the medical field, it is necessary to predict and diagnose the risk of infection: the quicker and more accurate the diagnosis, the more effective the management of the patients, the risk of transmission being minimized. The approach is similar for animal health.
There are identical problems in the food industry. There, however, a distinction is made between:                pathogenic microorganisms and their toxins, where research applies to the raw materials, intermediates, and marketed finished products,        non-pathogenic microorganisms, used as indicators of quality of the production process, from the raw materials to the finished products, throughout the chain, and        bacteria of technological interest such as ferments.        
Rapid and precise detection of suspected contaminants makes it possible to control them and thus apply corrective measures.
Technically, microbiological analysis can employ one or more steps of pre-enrichment and/or enrichment, one or more steps of detection, and one or more steps of counting the microorganisms. For particular applications such as microbiological control in the food industry, a confirmation step may also be required, in order to comply with the standards in force in this field.
At present, no method exists for detecting a target microorganism in a large initial amount of sample, without employing an enrichment step.
The enrichment step employs selective or non-selective culture media, which aim to promote growth of the target microorganisms in biological or environmental samples, while limiting the growth of the non-target flora. The media are often used in containers of the sterile plastic bag type, in which they are brought into contact with the food samples or environmental samples, for purposes of resuspension and enrichment of the microorganisms being sought. This step is necessary in order to meet the requirement of detecting the potential initial presence of at least one target microorganism in an amount of sample that is very variable and optionally is very large, e.g. 25 grams (g) to 375 g diluted in 225 to 3375 milliliters (mL) in the culture medium. At the end of this enrichment step, an aliquot (from 5 microliters (μl) to 5 mL) is taken for carrying out the step of detecting the target microorganisms. Now, it is necessary for this aliquot to contain a sufficient amount of target microorganisms to ensure that they are systematically detected. A step of secondary enrichment or subculture may then be necessary.
The detection step is based historically on culturing the microorganisms on agar media, for detecting the metabolic characters of the microorganisms being sought. Conventionally, specific enzymatic substrates are used. These enzymatic substrates generally consist of two parts, a first part specific to the enzyme activity to be detected, also called the target part, and a second part acting as a marker, called the marker part, generally consisting of a chromophore or a fluorophore. Based on the choice of these substrates, depending on whether there is reaction or not, it is possible to characterize the nature of a microorganism or distinguish between different groups of microorganisms. Thus, appearance or disappearance of coloration or of fluorescence will be the signature of a genus or of a type of microorganism. In this respect, the use of chromogenic media allows simultaneous detection and identification of the microbes being sought. It simplifies the process and greatly reduces the time to obtain the result. We may mention, as a concrete example, the applicant's ChromID® media. These chromogenic media are based on detection of specific metabolic characters of the microbes being sought, for example beta-glucuronidase enzyme activity for Escherichia coli. 
Immuno-assays constitute another of the technologies used for detection testing. They make use of the immunogenic characteristics of the microorganisms being sought. Non-exhaustively, we may mention the techniques of immunofluorescence, the ELISA (Enzyme-Linked ImmunoSorbent Assay) techniques, competitive or of the sandwich type. These techniques employ a step of so-called indirect detection that employs a secondary antibody conjugated with an enzyme for subsequent detection via a substrate specific to the latter.
Document EP-B-I 440 316 describes for example a device for detecting microorganisms. This device consists of a solid substrate, on which capture partners specific to the target microorganisms, such as antibodies, are fixed. The capture substrate is then placed in various containers comprising the sample to be analysed and the various reagents for carrying out an ELISA reaction.
This step of so-called indirect detection then involves (following the enrichment step) the execution of various treatment steps (taking the sample, heating, centrifugation, washing, etc.) of the sample before the screening/detection step, which consequently make the operating protocol more complex, make the analysis less convenient and increase the time to supply the results.
Finally, the techniques of molecular biology, based on the genomic characters of the microorganisms being sought, are also employed for detecting and identifying the target microorganisms. We may mention, as examples, the conventional techniques of amplification such as PCR (Polymerase Chain Reaction) and NASBA (Nucleic Acid Sequence Based Amplification), which can be coupled to techniques for real-time detection known by a person skilled in the art. Nevertheless, these techniques require an arduous step of preparation of the samples, consisting of isolating the microorganisms, lysing them in order to release the nucleic acids, and finally purifying the latter. This also has a direct effect on the complexity of the operating protocol, making the analysis less convenient and increasing the time to supply the results.
Regarding the confirmation step, it is more particularly associated with microbiological analysis in the food industry. In fact, when the result of the methods developed previously is positive, it is necessary to confirm the presence of the pathogen being sought. This requires an additional test and the use of a principle of detection different from that used in the first analysis. The techniques described above are used at leisure for confirmation.
The complete and accurate identification of a microorganism in a sample therefore requires several successive steps: enrichment, optionally subculture, detection and confirmation. Standardization of the tests used routinely has allowed automation of the methods of detection, but they still take a long time. A drawback of the prior art is in fact that these steps are carried out sequentially and require a large number of time-consuming manipulations, thus having an impact on the time taken to supply the results.